a) Field of the Invention
The invention is directed to a device for optical analytic measurement in a multisample carrier, particularly for measuring fluorescence or bioluminescence, in which a plurality of samples are read out simultaneously for optical analysis in addition to the reaction process and excitation process. The invention is preferably applicable for analysis of biochemical or cell-based assays which require dispensing liquids into wells of the multisample carrier together with optical excitation and simultaneous readout of the samples.
b) Description of the Related Art
Particularly in biochemistry and pharmacology, it is necessary to test as many different substances as possible in microtitration plates (so-called multiwell plates or microplates as they will be referred to hereinafter for the sake of brevity) in combination with reagents and/or cells within a short period of time. This is usually carried out in the form of an assay in which it is precisely determined in what sequence and at what time the microplate with its samples must be at what location. Often, the reactions of living cells on substances of pharmacological interest are tested. For this purpose, the cells must be kept in a nutrient medium at a specified temperature and mixed with substances, kept in the incubator again for a defined period of time, and so forth. But the reverse is also possible; namely, substances are added to the wells of the microplate which hold reagents or cells.
In many cases, this preparatory handling concludes with the measurement of optical signals. For this purpose, one or more reagents are added to the cells before or during the measurement of light. Liquid is added to as many (or all) of the wells of the microplate as possible and, further, the light emission is measured simultaneously starting with the addition of liquid. In this connection, there are many competing demands when high microplate throughput is to be achieved with automatic HTS (High Throughput Screening) or UHTS (Ultra-High Throughput Screening). Since the generated light emission per well can often only be observed for a few seconds, a measurement of intensity per well with a time resolution in the range of seconds is required. However, the total measurement time over an entire microplate should be as short as possible.
Many different fluorescence measurement devices are known in the art, some of which are also outfitted with integrated liquid dispensers (so-called liquid handling).
For example, U.S. Pat. No. 6,372,183 describes a fluorescence measuring device in which liquid is dispensed. The liquid is dispensed in individual, preselected wells by means of an individual dispenser or by means of pipettes arranged in a row in order to dispense liquid simultaneously in a column of the microplate. The dispensing position is located before the measurement position spatially so that it is necessary to displace the microplate after adding liquid in order to measure the well in which the liquid has been dispensed; therefore, a time delay occurs. Accordingly, the microplate must be moved in order to dispense into additional wells, while the wells to which liquid has been added previously are measured simultaneously by a photon counter (PMT). For fluorescence measurement, a CCD array is described as detector; however, this may also be only a line counter, but the type and manner of optical imaging of the microplate is not mentioned in this regard. The measurement of the microplate in a plurality of wavelengths of the excitation light is carried out sequentially by adjusting a filter wheel. However, a procedure of this kind requires switching intervals of several seconds each and is not suited to HTS.
US 2001/0028510 describes a fluorescence arrangement in epi mode, as it is called, in which the excitation and emission are carried out coaxial to and perpendicular to the microplate. Also, an objective is described which is used for excitation and for recording fluorescence. Further, a ring light is focused on the object by a front lens of the objective. The disadvantage in this arrangement consists in that there is no separation of the excitation beam from the readout beam, i.e., tube brightening exists due to the shared use of the front lens and generates a considerable proportion of the background light. Also, WO 02/068942 discloses a fluorescence measuring device for time-resolved fluorescence measurements in which a beam splitter is used between the objective and the front lens and which therefore has the same disadvantage.
Similarly, WO 01/04608 describes a light detection device for different spectroscopic examinations (including fluorescence intensity, fluorescence duration and fluorescence polarization, etc.) in which the illumination and detection beam path, which is likewise unitary, is collimated and guided parallel to the microplate and a sensitive positioning of the illumination and detection on the individual wells is carried out by means of a movable focusing deflecting mirror. Troublesome tube brightening and background fluorescence from the supernatant sample are also inevitable in this case.
WO 01/01112 discloses an epi mode construction with a beam splitter (tube brightening), an array of microobjectives for individual illumination of every individual well, and a CCD camera. The disadvantage of the system is that there is a separate microobjective for each well so that when the microplate format changes (with an increased quantity of wells) the system malfunctions or special masks which disadvantageously limit the illumination efficiency would have to be used. The microlens array is again used to image the wells on the CCD camera with similar disadvantages (quantity of channels and quantity of pixels of the CCD).
U.S. Pat. No. 5,355,215 discloses an instrument that specifically reduces the unwanted background fluorescence of the well liquid and accordingly improves the wanted signal from a cell layer at the (transparent) base of the wells. The excitation light source impinges on the base of the microplate at an oblique incident angle from below and, in addition, the light bundle cross section per well is limited through a multi-pinhole diaphragm in order to observe the fluorescent radiation, as far as possible, only from a small section volume at the base of each well. As a general condition, an optical illumination axis or detection axis is directed at an angle to the normal direction of the microplate. However, due to the sharp divergence of the excitation bundle, the excited liquid volume in the wells is dependent upon position. This dependency on angle also occurs when a laser is used, as is also mentioned in this reference.
The problem of interfering background fluorescence is also addressed in U.S. Pat. No. 6,420,183, where an absorption dye is added to the supernatant solution and eliminates the exciting beam and the emitted radiation in the supernatant liquid over the cell layer to be observed. However, the use of absorbent dyes is also problematic because, on the one hand, their biochemical reaction is unclear and, on the other hand, the absorption in the centrate is incomplete and can ultimately also have an (undesirable) effect in the cell layer.
Another publication (DE 197 55 187 A1) describes a sequential method that simultaneously adds liquid to a well and excites and measures fluorescence. A lightguide and photomultiplier are used. The lightguide is arranged orthogonal to the sample carrier and the excitation and reagent injection are carried out at oblique angles. The sample carrier is likewise moved in order to assist in dispensing and measuring individually (or by column).